Effective chemiluminogenic systems based on acridinium esters bearing substituents of various electronic and steric properties

Article abstract of DOI:10.1039/c5ob01798j

A series of 10-methyl-9-(phenoxycarbonyl)acridinium trifluoromethanesulfonates (XAEs), bearing substituents of various characteristics in the lateral benzene ring (2-halogen, 2,6-dihalogen, 2-trifluoromethyl, 2-nitro, 2-methoxy, 3-halogen and 4-halogen) w

Full text of DOI:10.1039/c5ob01798j

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Effective chemiluminogenic systems based on
acridinium esters bearing substituents of various
electronic and steric properties†
Cite this: DOI: 10.1039/c5ob01798j
Beata Zadykowicz,a Justyna Czechowska,a Agnieszka Ożóg,a Anton Renkevichb and
Karol Krzymiński*a
A series of 10-methyl-9-(phenoxycarbonyl)acridinium trifluoromethanesulfonates (XAEs), bearing substi-
tuents of various characteristics in the lateral benzene ring (2-halogen, 2,6-dihalogen, 2-trifluoromethyl,
2-nitro, 2-methoxy, 3-halogen and 4-halogen) were synthesized with high yields, identified and subjected
to a physicochemical and theoretical investigation. The main task of the work was to assess the mechan-
ism and optimal conditions of light emission in various liquid systems based on the above salts in order to
evaluate their potential usefulness as chemiluminescence (CL) labels and indicators in ultra-sensitive ana-
lyses. Density functional theory (DFT) calculations were performed to investigate the detailed mechanism
of the oxidation of 9-substituted 10-methylacridinium cations involved in XAEs by hydrogen peroxide in
alkaline media. Three general pathways were drawn, which are termed the “light path” (chemiluminogenic)
and there were two “dark paths” (non-chemiluminogenic): hydrolytic and “pseudobase”. The CL time
profiles, triggered in alkaline solutions containing hydrogen peroxide, enabled us to establish crucial
physicochemical parameters, including pseudo-first order kinetic constants of CL decay and relative
efficiencies of emission. In order to optimize the systems’ luminogenic performance, different bases, such
as sodium hydroxide, tetrabutylammonium hydroxide (TBAOH) and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU), as well as enhancers, such as cationic, zwitterionic and neutral surfactants (cetyltrimethyl-
ammonium chloride (CTAC), N,N-dimethyldodecylammonio-1,3-propane sulfonate (DDAPS) and Triton
X-100, respectively) were tested. The results revealed the optimal CL systems, which enabled us to obtain
substantially higher emissions than typical ones, based on acridinium esters or luminol. The derived para-
meters, characterizing the potential utility of the acridinium esters, such as stability in aqueous environ-
ments and usefulness (the product of emission efficiency and stability at a given pH), enabled us to reveal
the best candidates and their practical applications. The post-reaction mixtures, analyzed by means of
chromatography (RP-HPLC) and mass spectrometry (ESI-MS), allowed us to verify the occurrence and
population of the products that were theoretically predicted, i.e. 10-methyl-9-acridinone (NMAON),
10-methylacridinium-9-carboxylic acid (NMACA) and substituted phenols (RPhOHs).
Received 27th August 2015,
Accepted 28th October 2015
DOI: 10.1039/c5ob01798j
www.rsc.org/obc
a high temporary intensity of emission – up to 100-fold higher
Introduction
than luminol.3 The quantum yields of most AEs are at the level
Chemiluminogenic acridinium esters (AE), first described by of 2% in aqueous environments, which can be considered a
F. McCapra in the 1960s, were introduced in medical diagnos- high value compared to luminol (ϕCL = 1.23%, the standard
tics about twenty years later.1,2 In contrast to the commonly used in luminometric assays4) enabling the direct quantifi-
employed phthalic acid hydrazides, they do not require the use cation of the analyte in a given system at the femtomolar or
of a catalyst to trigger CL and, in general, are characterized by even lower levels of concentrations.5,6 The carriers of AEs and
some other quaternary acridinium salts (9-sulphonamides,
9-cyanides) in modern analysis are also determined by the
aFaculty of Chemistry, University of Gdańsk, Wita Stwosza 63 St., 80-308 Gdańsk,
Poland. E-mail: karol.krzyminski@ug.edu.pl
possibility of their relatively easy chemical modifications and
the simplicity of the CL assays.7,8 Today, applications of AEs
bDepartment of Chemical Metrology, Kharkiv V.N. Karazin National University,
cover a wide area of immunodiagnostics, where they are used
Kharkiv, 61022, Ukraine
in a chemically bound form (CL labels),8 as well as in environ-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob01798j
mental analyses – where they are utilized in the free form
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(CL indicators).6,9 Acridinium labels and indicators have been an important observation for the rational design of new acri-
employed, among others, for assaying human α-fetoprotein, dine-based labels and indicators.
gonadotropins, thyroid stimulating hormone (TSH), viruses,
Natrajan’s group determined the influence of various types
markers of cancer cells, nucleic acids, oxidative enzymes (e.g. of surfactants on the behavior of zwitterionic and alkyl-substi-
glucose oxidase, horseradish peroxidase) and other important tuted AE labels and their protein conjugates.24
bioanalytes.1–3,8,10,11 AEs were also utilized to determine trace
To take a deeper look at the physicochemistry and lumino-
amounts of environmental pollutants, such as hydrogen per- genic properties of the AEs, we have synthesized a series of
oxide or substituted phenols.3,12 Recently, acridinium salts chemiluminogenic salts, differing in terms of the location and
were employed in serial clinical assays performed with the aid the electronic and steric properties of the substituent(s) intro-
of automated clinical analyzers – such as Abbott Prism® or duced into the lateral benzene ring (XAEs). In the past, we per-
Siemens Advia Centaur® – in the routine monitoring of blood formed studies on the crystalline structures of XAEs, applying
quality for the presence of HBC, HCV, HIV-1, HIV-2 and other X-ray crystallography methods, as they demonstrate interesting
viruses.10 Other examples of diagnostic methods developed features in the solid phase.25–31 On the other hand, applying
with the participation of AEs are affinity tests, applied in the differential scanning calorimetry methods to study their
investigation of nucleic acids (also in automated variants).13,14 thermal behavior enabled us to assess important physico-
The latter approach makes an alternative for DNA sequencing, chemical parameters – such as the crystal lattice energy,
gel electrophoresis, mass spectrometry and other techniques enthalpies of volatilization and formation and others.32
employed in nucleic acid chemistry. Recently, new variants of
In this work, we present investigations of the luminogenic
nucleic acid hybridization AE-based tests, with no need for properties of XAEs, including theoretical, structural and
their amplification, were proposed.15,16 The introduction of physicochemical studies on the liquid phase. Since most of
the electron-donating alkoxy substituents (e.g. OCH3) into the the compounds presented here express higher emission
conjugated ring system of AE resulted in a shift of the emis- efficiencies than their alkyl analogues,22 we attempt to reveal
sion range towards longer wavelengths, and a slight enhance- the possible reason(s) for this phenomenon, including ana-
ment of its efficiency.16,17 The authors also stated that the lyses of the post-CL reaction mixtures. Within this work we
introduction into the benzene ring electron-withdrawing Br also took into consideration the emission properties and
atoms (ortho positions) of AE enables the triggering of the ring crucial analytical parameters of XAEs in systems containing
efficient emission at relatively low pH values (pH = 9). The pro- cationic, zwitterionic or neutral surfactants, being employed as
posed design of new AE labels should guarantee their CL enhancers in this context.24 In order to understand the
increased resistance to hydrolysis and good dynamics of light observed differences in physicochemical features, we applied
emission (which finally enables lower limits of detection). computational methods based on Density Functional Theory
Other studies on variously substituted acridinium esters indi- (DFT), which enabled us to reveal the thermodynamic and
cated that the introduction of oxygen-rich groups (such as kinetics parameters and to propose possible reaction pathways
polyethylene glycol (PEG) residues or alkylsulfopropyl groups) for chemiluminogenic oxidation of XAEs in various alkaline
and bulky polar substituents (such as Br) into specific environments.
locations of the aromatic ring systems results in a substantial
increase in the luminogenic capability of the respective com-
pounds (up to four times).18–21 The polar groups improve the
solubility of the related derivatives and, presumably, help to
reduce their hydrophobic aggregation after conjugation with
the bioanalyte accompanied with low non-specific binding.
Results and discussion
Origin of chemiluminescence
Previous investigations by us22 and others8,33 on the mechan-
Our previous investigations on the CL of AEs were focused ism of the oxidation of 9-substituted 10-methylacridinium
on the family of alkylphenyl esters of 10-methylacridinium-9- cations by H2O2 in alkaline media indicated that the primary
carboxylic acids – molecular systems frequently incorporated steps of the reaction of XAEs with either OOH− or OH− are
in commercially available CL labels.22,23 Experimentally estab- mainly determined by the location of the electrophilic center
lished values – CL decay kinetic constants and integral in the cation. According to the frontier orbital theory,34 the
efficiency of emission – showed correlations with the theoreti- Lowest Unoccupied Molecular Orbital (LUMO) distribution of
cally predicted parameters, such as activation barriers and an electrophilic species determines the molecular center sensi-
specified geometrical parameters of the molecular systems tive to nucleophilic attack. The value of the LCAO coefficient of
involved in the light-emitting step. The presence of bulky alkyl the pz atomic orbital in the XAEs’ LUMO is ca. 10 times higher
substituents at the ortho-positions of the benzene ring drasti- at the endocyclic C(9) than at the C(15) carbonyl atom (0.30
cally decreased the reaction course; their slight electron-donat- and 0.03, respectively; average for 2-F, 2-CF3, 2-NO2, 2-OCH3
ing character is manifested by the same effect, but more and 2,6-diBr) (Scheme 1). This implies that C(9) rather than
subtle. Among the interdependences revealed within this C(15) marks the site of the primary nucleophilic attack of the
family of CL systems was the correlation between the AE’s above-mentioned anions, despite the fact that the Mulliken
emission intensity and the volume of the hydration layer of the partial charge at C(9) is lower than at the C(15) atom of the
leaving group22 during the light-generating step. The latter is carbonyl group (0.09 and 0.52, respectively; average for 2-F, 2-CF3,
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Scheme 1 Structural formula and systematic names of the investigated 10-methyl-9-(phenoxycarbonyl)acridinium trifluoromethanesulfonates
(triflates) (XAEs) with the numbering system of C, N and O atoms given for the cations. The number of H atoms is the same as the C atoms to which
the former are attached.
2-NO2, 2-OCH3 and 2,6-diBr). Another premise that must be
Taking into account the reaction pathways proposed by
taken into account is the fact that H2O2 reacts spontaneously other authors8,33 and our previous investigations,22 as well as
with OH− to yield OOH− (the thermodynamic data for the reac- the information mentioned above, we considered the pro-
tion H2O2 + OH− = H2O + OOH− are: −26.8, −27.9 and cesses in accordance with general chemical knowledge that
−20.6 kcal mol−1 (enthalpy (gaseous phase), Gibbs’ free energy may explain the experimental findings. The pathways of the
(gaseous phase) and Gibbs’ free energy (aqueous phase) of the possible reactions of XAEs with OOH− and OH−, outlined in
reaction at temperature 298.15 K and standard pressure, Scheme 2, emerge from a thorough analysis of the results of
respectively).
theoretical calculations. Scheme 2 presents the processes that
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Scheme 2 Pathways of the reactions of MPCA+ with OOH− and OH− (pathway one (blue arrows), pathway two (red arrows) and pathway three
(green arrows).
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account for CL (pathway one; steps I, VI, and VII; or I, XVI and leading to the energy-rich 10-methyl-9-acridinone (c) (step VII).
XVIII), the formation of the so-called “pseudobase” (step II), Interestingly, in the case of cations involved in 2-NO2 and 2,6-
and the following non-CL processes (pathway two; steps IX diBr (Scheme 1), we did not observe steps VI and VII. After the
and X; or XI, XII and XIII), the appearance of 10-methyl- attack of OOH− on the carbon atom C(9) of 2-NO2 or 2,6-diBr
acridinium-9-carboxylate anions or 10-methylacridinium carb- cation (step I), followed by the reaction of the addition product
oxylic acid (pathway three; steps IV and XV, and step V, a with OH− (step VI), the elimination of an adequate number
respectively) among the reaction products. The theoretically of phenoxy anions and the resulting cyclic intermediate (e)
predicted thermodynamic data for all the reaction steps are (step XVI) with the subsequent elimination of carbon dioxide
given in Table 1, while Table 2 contains kinetic data for the leads to the energy-rich 10-methyl-9-acridinone (c) (step XVIII).
steps involving molecules in the ground electronic state. We examined the two pathways (steps I, VI and VII; and I, XVI
Additionally, the DFT-optimized geometries originating from and XVIII) for compounds 2-F, 2-CF3 and 2-OCH3. The theoreti-
the acridine molecules produced by the reaction of XAEs with cally predicted thermodynamic data indicated that these two
OOH− or OH− are shown in Fig. S1 (lowest energy and tran- pathways are possible for compounds 2-F, 2-CF3 and 2-OCH3.
sition state structures) in the ESI.†
On the other hand, the enthalpy (gaseous phase), Gibbs’ free
Pathway one (“light pathway”) represents the attack of energy (gaseous phase) and Gibbs’ free energy (aqueous
OOH− on the XAE acridinium carbon atom (9) (step I), fol- phase) of step XVI (298.15 K, standard pressure) are thermo-
lowed by the reaction of the addition product a with OH− (step dynamically more preferred in the case of compounds 2-NO2
VI), the cyclization of the anion to a cyclic intermediate (b) and 2,6-diBr than 2-F, 2-CF3 and 2-OCH3. The barriers pre-
(step VI), and the elimination of the phenyl carbonate anion dicted for step XVIII are similar in the aqueous phase to the
Table 1 Thermodynamic data of the elementary steps of the reactions of selected compounds with OOH− and OH− a
Gaseous phase
r,298H° r,298G°
Aqueous phase
r,298G°
Gaseous phase
Aqueous phase
r,298G°
Step no.
(Scheme 2)
Step no.
(Scheme 1)
Substituent
Δ
Δ
Δ
Substituent
Δr,298H°
Δr,298G°
Δ
I
2-F
−164.2
−167.1
−166.0
−162.8
−165.4
−190.2
−192.9
−191.4
−188.9
−188.0
−138.4
−142.0
−135.4
−133.9
−138.3
−156.6
−152.3
−75.4
−86.4
−96.3
−67.2
−91.7
−77.2
−80.1
−72.0
−93.6
−95.7
−97.6
10.8
−151.3
−153.2
−153.5
−150.5
−152.8
−180.2
−182.0
−181.1
−178.4
−178.5
−125.5
−128.2
−123.3
−121.0
−124.9
−146.6
−142.1
−78.0
−89.1
−99.8
−69.7
−94.8
−78.3
−81.9
−72.6
−108.0
−111.0
−112.0
2.2
−43.3
−44.1
−44.7
−43.0
−44.2
−63.1
−64.4
−64.9
−62.7
−62.6
−25.0
−26.0
−23.0
−22.4
−25.0
−36.7
−34.9
−51.7
−58.8
−68.4
−46.9
−64.7
−40.4
−42.7
−37.6
−117.7
−117.8
−118.0
0.4
X
2-F
2-CF3
2-NO2
2-OCH3
2,6-diBr
2-F
−0.1
−6.6
−24.4
−30.9
−38.5
−21.8
−44.2
−56.0
−58.0
−61.7
−50.9
−51.0
10.2
−34.4
−39.0
−47.6
−30.9
−45.8
−15.7
−16.8
−21.1
−12.2
−14.2
−3.7
2-CF3
2-NO2
2-OCH3
2,6-diBr
2-F
2-CF3
2-NO2
2-OCH3
2,6-diBr
2-F
2-CF3
2-NO2
2-OCH3
2,6-diBr
2-F
2-OCH3
2-F
2-CF3
2-NO2
2-OCH3
2,6-diBr
2-F
2-CF3
2-OCH3
2-F
2-CF3
2-OCH3
2-F
2-CF3
2-OCH3
2-F
−13.9
2.3
−20.2
−70.3
−72.6
−75.3
−64.4
−66.7
24.7
24.4
15.8
31.5
8.4
−94.2
−103.1
−105.3
−100.9
−88.4
−84.5
−66.3
−74.5
−85.4
−59.5
−81.5
−92.2
−99.6
−116.0
−88.4
−108.6
−93.7
II
III
XI
XII
2-CF3
2-NO2
2-OCH3
2,6-diBr
2-F
2-CF3
9.8
1.9
18.6
−6.3
−8.4
2-NO2
2-OCH3
2,6-diBr
−13.1
−2.7
−18.7
−118.6
−58.7
−60.8
−58.2
−82.9
−80.0
−51.6
−57.9
−66.8
−47.1
−63.7
−69.9
−76.0
−88.5
−67.6
−82.8
−106.2
IV
V
XIII
XIV
−116.3
−104.1
−106.9
−102.2
−103.0
−99.1
−80.1
−89.2
−99.6
−72.5
−95.4
−105.8
−114.2
−129.7
−102.0
−123.2
−104.1
2-F
2-CF3
2-OCH3
2-F
2-OCH3
2-F
2-CF3
2-NO2
2-OCH3
2,6-diBr
2-F
2-CF3
2-NO2
2-OCH3
2,6-diBr
XV
XVI
VI
VII
VIII
IX
XVII
7.7
16.4
−0.3
8.1
−3.6
2.4
−68.0
−69.8
−73.9
−63.5
−66.5
−68.7
−71.4
−75.4
−64.7
−67.4
−33.2
−34.5
−34.9
−32.3
−35.4
XVIII
2-CF3
2-NO2
2-OCH3
2,6-diBr
a Δr,298H° and Δr,298G° respectively represent the enthalpy and Gibbs’ free energy (gaseous phase) or free energy (aqueous phase) of the reaction
corresponding to a given step number at temperature 298.15 K and standard pressure. All thermodynamic data are in kcal mol−1
.
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Table 2 Kinetic characteristics of the elementary steps of the reaction of selected compounds with OOH− and OH− a
Gaseous phase
a,298H°
Aqueous phase
a,298G°
Step no.
(Scheme 2)
Substituent
Δ
Δ
a,298G°
298k° (298τ°99
)
Δ
VII
2-F
9.3
10.8
8.2
8.5
8.2
7.7
8.8
2.0
32.6
10.3
8.5
10.7
8.6
7.7
7.1
6.5
7.9
2.8
33.0
10.1
3.4 × 106 (1.4 × 10–6
9.5 × 104 (4.8 × 10–5
3.3 × 106 (1.4 × 10–6
3.4 × 106 (1.4 × 10–6
4.1 × 107 (1.1 × 10–7
9.8 × 107 (4.7 × 10–8
1.1 × 107 (4.3 × 10–7
)
)
)
)
)
)
)
14.1
14.6
12.6
14.1
8.2
6.6
8.0
5.2
26.2
15.4
2-CF3
2-OCH3
2-F
2-CF3
2-NO2
2-OCH3
2,6-diBr
X
5.4 × 1010 (8.5 × 10–11
4.0 × 10–12 (1.1 × 1012
)
)
XIII
XVIII
2.6 × 105 (1.8 × 10–5
)
a Δa,298H° and Δa,298G° (both in kcal mol−1) respectively represent the enthalpy and Gibbs’ free energy (gaseous phase) or free energy (aqueous
phase) of activation at temperature 298.15 K and standard pressure. 298k° (in s−1) and 298τ°99 (in s) (eqn. (5) and (6)) respectively denote the rate
constant and the time after which the reaction is 99% complete.
barriers predicted for step VII (e.g. for compound 2-F Δa,298G° however, exist for the final steps (X and XIII), characterizing
in the aqueous phase (in kcal mol−1) are 15.4 and 14.1, the transformation of the XAE pseudobase form to 10-methyl-
respectively). The computationally predicted pathways of CL 9-acridinone (Table 2). In the case of step X, the activation bar-
generation do not clearly present which pathway operates for riers fall in the range of 5–14 kcal mol−1, which is comparable
the cations involved in 2-F, 2-CF3 and 2-OCH3. Just one to the activation barriers characterizing step VII. Nevertheless,
thermodynamically possible pathway was observed in the case the energy released is too small to electronically excite the
of XAEs, where the elimination of phenoxy anions after the above product (Gibbs’ free energy in the aqueous phase is
reaction of the addition product a with OH− (step VI) and for- between −30 and −47 kcal mol−1) (Table 1). The overall values
mation of the cyclic intermediate e occur. The energy released of the thermodynamic functions for step XIII (Table 1) are
in step VII or XVIII (Table 1) exceeds that which is necessary to high enough to ensure electronic excitation of the produced
cause the electronic excitation of 10-methyl-9-acridinone, 10-methyl-9-acridinone, but the activation barrier characteriz-
which is 88.2 kcal mol−1 35
.
ing this process is significantly higher than that of step VII or
Pathway two (“dark pathway”) assumes the attack of OH− X (Table 2). For the above reasons, pathway two, assuming the
on the XAE carbon atom (9) (step II), followed by the reaction attack of OH− on XAEs’ C(9) cannot lead to CL, represents the
of the addition product f (‘pseudobase’) with OH− (step IX) dark transformation of XAE cations, producing 10-methyl-9-
and the elimination of an adequate number of phenoxy anions acridinone in the ground electronic state.
and carbon monoxide, leading to 10-methyl-9-acridinone (c)
Pathway three explains the occurrence of 10-methyl-
(step X). Alternatively, the reaction of the addition product f acridinium-9-carboxylate anions or 10-methylacridinium
(‘pseudobase’) through the attack of OOH− on the carbon 9-carboxylic acid (steps IV and XV, and step V, respectively) among
atom C(15) of the carbonyl group of the XAE (step XI), followed the reaction products, as OH− – like OOH− – can attack the
by elimination of a phenoxy anion leads to intermediate i (step acridinic carbon atom (9) or the carbonyl carbon atom (15).
XII), and the further elimination of carbon dioxide and water The addition of OH− to C(9) (pathway two) is more probable
leads to 10-methyl-9-acridinone (c) (step XIII). The theoretically for the reasons mentioned above. As we noted earlier,22 the
predicted thermodynamic data indicated that these pathways identified entities were 10-methyl-9-acridinone (NMAON),
are possible for all of the investigated compounds. The 10-methylacridinium 9-carboxylic acid (NMACA) and substituted
enthalpy (gaseous phase), Gibbs’ free energy (gaseous phase) phenols (RPhOHs). The latter acid or its zwitterionic form,
and Gibbs’ free energy (aqueous phase) computed at tempera- 10-methylacridinium-9-carboxylate, is likely formed as a result
ture 298.15 K and standard pressure suggest that the addition of XAEs’ hydrolysis, which assumes the attack of OH− on the
of OH− to C(9) (step II) is thermodynamically preferred over carbonyl carbon C(15) (step IV), followed by the reaction of the
the addition of OOH− to the same carbon atom. In this case, addition product j with OH− (step XV), and the elimination of
the possible pathway of the reaction seems to be exclusively an adequate number of phenoxy anions and water molecules,
dependent on the kinetic factors. We noted earlier,22 and have leading to product (k). These two steps are possible only for
confirmed in our present calculations, that the activation bar- the two species
–
9-[(2-fluorophenoxy)carbonyl]-10-methyl-
9-[(2-methoxyphenoxy)-carbonyl]-
riers characterizing the addition of OOH− or OH− to C(9) of acridinium (2-F) and
the acridinium esters (steps I and II, respectively) and the sub- 10-methylacridinium (2-OCH3) cations. In other cases (2-CF3,
stitution at OOH− of the carbonyl C(15) atom of the ‘pseudo- 2-NO2 and 2,6-diBr), these steps presumably do not occur and
base’, followed by the release of the phenoxy anion (steps XI NMACA is produced upon the attack of OH− on the carbonyl
and XII, respectively) are negligible. Activation barriers do, carbon atom C(15) (step V), followed by simultaneous
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elimination of the substituted phenoxy anion leading to Spectroscopic properties of XAEs
product (l). The thermodynamic data (Table 1) indicate that
this step is possible for all the theoretically investigated com-
pounds that are 2-F, 2-CF3, 2-NO2, 2-OCH3 and 2,6-diBr. In the
case of step V the alkaline hydrolysis of acridinium esters
occurs with the formation of a tetrahedral intermediate, which
immediately decays into products (l) and the substituted
phenoxy anion. The activation barriers characterizing the
addition of OOH− to the C(15) atom and formation of a tetra-
hedral transition state are negligible (0.6 kcal mol−1; average
value for 2-CF3, 2-NO2 and 2,6-diBr). In our opinion the more
preferred pathway is via the steps IV and XV, but the theoreti-
cally predicted data indicated that the latter pathway is not
possible in the case of 2-CF3, 2-NO2 and 2,6-diBr.
1
According to H and 13C NMR techniques that were employed
for the identification of cations involved in XAEs, the compat-
ibility of the experimental and computational chemical shifts
was satisfactory as denoted by the data collected in Tables S1–
S3 and illustrated in Fig. S2 (ESI†).
1
In general, the H NMR spectra of XAEs can be subdivided
into three groups of signals37 – those arising from the hydro-
gen atoms bonded to the acridine/acridinium nuclei
(maximum range of chemical shifts, σ), the hydrogens present
in the benzene ring (an intermediate range of σ), and – if
present – the signals rising from the aliphatic hydrogen atoms
(the lowest range of σ). In the group of 9-[(phenoxy)carbonyl]-
acridines (the unmethylated precursors of XAEs), the 1H
signals from the acridine nucleus H1/H8 and H4/H5 always
occur at higher values of chemical shifts than H2/H7 and H3/
H6 (Fig. S2, ESI†). The latter observation is likely related to the
“uncover” effect, applied by the carbonyl oxygen atom (in the
case of H1/H8) or the endocyclic nitrogen atom (in the case of
H4/H5). Due to their spatial proximity, the H1/H8 signals also
seem to be most sensitive to the substitution type in the
Synthetic considerations
Although the methods for the preparation of 10-substituted
9-(phenoxycarbonyl)acridinium salts have been described in the
literature,22,25–32,36 a few remarks concerning the compounds
investigated here should be made (their synthesis and spectro-
scopic analyses are reported in the ESI†). To ensure a clean
and effective synthesis of XAEs (Scheme 1), their immediate
precursors, substituted 9-[(phenoxy)carbonyl]acridines, must
be obtained in high yield and purity. According to this, we
promptly chromatographed the reaction mixtures after the
completion of the reaction, omitting numerous washings of
the organic post-reaction mixture with aqueous solutions that
lower the yield of reaction.22 The procedure is especially
important if the sterically hindered phenols are used for esteri-
fication or the product contains hydrophilic atoms or groups
of atoms. TLC tests of the chromatographically (LC) purified
(9-[(phenoxy)carbonyl]acridines showed no other residual acri-
dines by fluorescence detection; however, the elemental ana-
lyses are not always satisfactory. Further investigation revealed
that the residual impurities in the chromatographed materials
arose from residual phenols, characterized by a similar polarity
as the uncharged esters. However, such impurities do not
affect the quality of the final products (XAEs) – as phenols are
washed away during the last step of the reaction (quaterniza-
tion). If needed, the residual phenols can be easily removed
from the final products by heating the products in a vacuum
to about 100 °C – under such conditions their sublimation/
evaporation takes place.
1
benzene ring. The H NMR spectra of XAEs are similar to the
spectra of their precursors (the synthesis of XAEs, ESI†). In
these cases, the signals rising from the acridinium nucleus
appear at higher value chemical shifts than the respective
signals for the bases – due to the lowering of the electron
density, especially in a spatial area close to the endocyclic
nitrogen atom. The coupling constants, characterizing the
direct vicinal protons in the XAEs, appear in a range of values
characteristic for aromatic molecular systems (7.0–10.0 Hz). In
the case of couplings of higher orders, the values are located
in the range of 0.5–2.5 Hz and reflect the individual features of
the analyzed molecular systems. The 13C NMR spectra of XAEs
(Fig. S2, ESI†) express a typical pattern, characteristic for
phenyl-substituted acridinium esters.37 Each of the observed
signal was attributed to the respective carbon atom (Table S2,
ESI†) with the aid of HETCOR H–13C HSQC two-dimensional
1
NMR spectra and the ACD/CNMR database.55
The data concerning cations involved in XAEs disclosed by
mass spectrometry are summarized in Table S1 (columns 1–3
from the right, ESI†) and the representative MALDI-TOF
spectra are presented in Fig. S2 (ESI†). In all cases, the mass
spectra present a simple and clear pattern, underlining the
chemical identity of the obtained acridinium salts. The parent
ions M+ or (M + 1)+/(M − 1)+ are observed with the relative
abundances of 18–100% and 8–76%, respectively and the
characteristic signal for all spectra – 10-methylacridinium
cation (MeAcr+, m/z ≈ 193, or (MeAcr+ + 1)+, (MeAcr+ − 1)+
The other important synthetic issue that must be remarked
upon in the reaction of the above-described esters with methyl
triflate is the special treatment of the solvent before the reac-
tion (chloroform should be passed through a dried, basic
Al2O3 column), as well as the need for the use of a “proton
sponge” (such as immobilized 2,6-di-tert-butylpyridine) – in
order to remove the acidic impurities from the reaction mix-
tures, greatly reducing the yield and quality of the obtained
salts. It must also be noted that, among other XAEs, the com-
pound encoded 2,6-diI (Scheme 1) caused experimental
signals)
21–100%.
– are observed within relative abundances of
Chemiluminogenic properties of XAEs
Kinetics of CL decay in various environments. The group of
difficulties as it expressed limited photochemical stability, the presently studied acridinium esters (XAEs) contains substi-
which was observed during the experiments.
tuents of various electronic and steric characters in the ester
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Fig. 2 CL parameters of XAEs (Scheme 1) in various environments. (A)
Pseudo-first order emission decay kinetic constants (kCL); (B) relative CL
efficiencies (RCLE, normalized) in various environments. The coefficients
of variability (CV) fall in the range of 2–8%. Abbreviations: TBAOH –
Fig. 1 Chemiluminescence profiles recorded for 2-substituted XAEs
(Scheme 1) using a standard triggering system. (A) CL profiles of 2-halo-
substituted aclidinium salts; (B) CL profiles of other 2-substituted acridi-
nium salts investigated. For details, see the Experimental section and
note 1 to Table S4 (ESI†).
tetra-n-butylammonium hydroxide, DBU
–
1,8-diazabicyclo[5.4.0]
undec-7-ene; MeOH – methanol. For details, see the Experimental
section and notes to Table S4 (ESI†).
tinct.21,39 The use of organic bases instead of NaOH – such as
sterically hindered tetrabutylammonium hydroxide (TBAOH)
or a strong Lewis base, 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in aqueous or methanolic environments was tested in
order to examine how it affects the kinetics and efficiency of
CL. We assumed that, with the use of other bases, the suscepti-
bility of XAEs to the production of undesirable “pseudobases”,
as well as the mobility of the ions engaged in the reaction,
might be altered. When using TBAOH, a clear acceleration of
the emission dynamics was observed if compared to the situ-
ation when typical triggers were used – by 20–50% in aqueous
and by 50–80% in methanolic environments (Fig. 2A; Table S4,
columns 3 and 5†). In turn, the use of DBU, in general, mani-
fests a decrease of kCL values by about 10–40% (Fig. 2A;
Table S4, column 4, ESI†). Such observations may be associ-
ated with a lower degree of H2O2 ionization in the environ-
ment when DBU was used instead of the typical Brønsted
bases. In addition, OH− ions also act as a catalyst in stage VI of
light-generating transformations (Scheme 2). Another obser-
vation, which might be annotated, is that the properties of the
environment (the base, solvent) express a more pronounced
influence on the emission kinetics in the group of 2,6-disubsti-
tuted than 2-substituted XAEs (Fig. 2A).
moiety (the leaving group during their chemiluminogenic oxi-
dation) located at the ortho, para or meta positions of the
benzene ring (Scheme 1). These substituents are electron-
acceptor halogen atoms, nitro (NO2) and trifluoromethyl (CF3)
and methoxy groups (OCH3), expressing an electron-donor
character.17,32
As shown by the data presented in Fig. 1 and 2A and sum-
marized in Table S4, column 2, ESI†), emission decays for
most haloester salts, initiated by standard triggering reagents
(H2O2/NaOH), represent high-speed (flash type) kinetics, with
kCL values falling in the range of ca. 3–5 s−1. Thus, the investi-
gated systems are characterized by emissions occurring in a
time order of seconds as shown in an exemplary CL time
profile for a series of 2-substituted XAEs (Fig. 1). The behavior
of the salts 2-NO2 and 2-CF3 is similar to the other 2-substi-
tuted haloesters. However, the compound bearing an electron-
donor substituent (2-OCH3) behaves differently in this context,
exhibiting a glow-type emission (kCL ≈ 0.4 s−1), typical for the
majority of previously studied acridinium alkylesters.17,32,38
Interestingly, the influence of the steric hindrance of substitu-
ents on kCL is not clearly observed, if one compares the kCL
values, e.g. for compounds 2-F/2,6-diF or 2-Br/2,6-diBr (Fig. 2A,
Table S4, ESI†). The latter behavior differentiates XAEs
(instead of 2-OCH3) from the above-mentioned alkylesters,
where the spatial effect caused by the ortho- substituents is dis-
Emission efficiency in various environments. The normal-
ized values of the relative efficiencies of XAE emission (RCLE)
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are compared in Fig. 2B and summarized in Table S4
(columns 6–9, ESI†). One can observe that, among the various
compounds investigated, the differences in RCLE are not sub-
stantial, as was observed in the case of the above-discussed
values of kCL. However, if one takes into consideration the
values of this parameter in various environments, the differ-
ences are more distinct.
Generally, the highest values of RCLE are obtained in
TBAOH/H2O, while the lowest values are obtained in the
TBAOH/CH3OH environment; the typical aqueous environ-
ment (NaOH/H2O) is characterized by moderate efficiency –
located in between the two former ones. Comparing the two
graphs presented in Fig. 2(A and B), it can be concluded that,
in aqueous systems,
a
faster emission is generally
accompanied by a higher efficiency. To some extent, this obser-
vation might be explained in terms of various amounts of
10-methylacridinium-9-carboxylic acid (NMACA), detected
among CL reaction products.22 The latter finding indicates
various levels of participation of the hydrolytic dark paths IV
and V (Scheme 2) among the reaction pathways. This problem
will be further discussed below (the products secreted upon
XAEs’ chemiluminescence). On the other hand – faster kinetics
in methanol is not accompanied by a higher efficiency emis-
sion of XAEs – as RCLEs characterizing the TBAOH/MeOH
system take the lowest values (instead of 2-OCH3, Fig. 2B,
Table S4 (ESI†)). The circumstances, whose importance is hard
to estimate just on the basis of CL measurements – such as an
altered mechanism of light generation in an organic environ-
ment or different excitation efficiency of the oxidation product
– might be considered to explain that observation.
The values of quantum yields (QY) determined for XAEs,
using luminol as a reference,22,40 are collected in column 10 of
Table S4 (ESI†). Generally, slightly higher emission yields for
XAEs than for the previously investigated acridinium alkyles-
ters in an alkaline aqueous environment (NaOH/H2O) can be
noted.22 In this context, the fluorinated derivatives are charac-
terized by maximal QY values in the group of investigated
salts, e.g. 2.3–2.4% in the case of 2-F and 3-F – which is more
than 25% higher than those obtained for the unsubstituted AE
in the same environment.
Fig. 3 CL parameters of XAEs (Scheme 1) in aqueous environments of
different alkalinity. (A) The dependence of RCLE (normalized to the
highest value) on the pH. (B) Usefulness (the product of stability at pH =
8.0 and the emission efficiency at given pH, normalized) in aqueous
environments of different alkalinity. The coefficients of variability (CV)
fall in the range of 2–8%. For details, see the Experimental section and
notes to Table S4 (ESI†).
reasonably well (about 50% of the original value) – such as
compounds 2,6-diF and 2-NO2. The latter effect is likely
associated with their substituent-driven activation towards
nucleophile attack (OOH−) onto the C(9) atom, starting
the “light pathway” of the oxidation of XAEs (Scheme 2,
pathway I).
Alkalinity of environment vs. CL efficiency. As regards the
influence of the environment’s pH on the emission efficiency
(RCLE) in the group of XAEs, the information obtained from
CL measurements is compared in Fig. 3A and summarized in
Table S4 (columns 11–13, ESI†). In general, a lower pH of the
CL environment is manifested by a substantial reduction in
RCLE values. This behavior is likely accompanied by a lower
population of OOH− ions (pKa of H2O2 = 11.75)41 in solutions
of lower alkalinity. The decrease in efficiency is irregular – a
change of pH from 12 to 10 leads to a slight (few percent)
reduction in the RCLE (as it takes place in the case of 2- and
4-substituted XAEs), whereas a further lowering of the pH
value (pH = 8) is manifested in a substantial reduction of
emission efficiency (up to several dozen percent). In the case
of 2-OCH3 the emission practically disappears at pH = 8.
However, some XAEs retain their emission ability at pH = 8
Other parameters characterizing XAEs’ emission ability. The
last column in Table S4 (ESI†) contains parameters named
stability, reflecting the degree of the retained XAE emission
activity after incubation in alkaline phosphate buffer (pH =
8.0, t = 30 min, T = 298 K). This parameter seems to be impor-
tant in the context of the utilization of XAEs as fragments of
CL labels – where the covalent attachment of a label to a bio-
molecule requires the incubation of the ester under the above-
mentioned conditions.8 As revealed from the data presented in
Table S4,† the stability of most XAEs fluctuates around 50%,
although some compounds (2-OCH3, 2,6-diBr) retain more
than 70% of their emission ability under experimental con-
ditions. The possible reasons for this might be considered in
the context of the electronic and steric influence of the substi-
tuents that are present in the benzene ring. In the group of
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2,6-dihaloesters, the trend is towards strengthening the com-
pounds’ stability in the order: 2,6-diBr > 2,6-diCl > 2,6-diF. The
least stable compound, that is 2-NO2, loses ca. 75% of its emis-
sion ability under the same conditions – apparently because of
its strong susceptibility to alkaline hydrolysis.
Interesting conclusions can be drawn from the chart, pre-
senting the “usefulness” of XAEs (Fig. 3B). Usefulness is the
original parameter, which we had proposed previously,22 as
the product of emission efficiency at a given pH (12 or 8) and
the stability in an alkaline environment (pH = 8). It can be
employed – among other physicochemical characteristics,
such as kinetics of emission, thermodynamic stability – as a
measure of potential utility of a given compound as a CL label
or an indicator. According to this, the compounds that are
intended to be used as CL labels should be characterized by
high usefulness at high pH values – under which the emission
Fig. 4 The interdependencies between estimated ionization constants
for respective phenols (pKa) [ACD55
]
and the pseudo-first order
constants of emission decay (kCL) (Table S4, column 2, ESI†) for 2- or
of the labeled conjugates is triggered. Accordingly, mono- 4-substituted XAEs (Scheme 1). The lines correspond to the following
equations: kCL = −2.593pKa + 26.44 (2-subst. XAEs), kCL = −2.045pKa
23.31 (4-subst. XAEs). For details, see the Experimental section and
note 1 to Table S4 (ESI†).
+
fluorinated compounds, such as 3-F, 2-F, 4-F or 4-Br and
2-OCH3, seem to be particularly useful in this context (Fig. 3B,
dark bars). On the other hand, if relatively low alkalinity, at
which the emission is triggered is important – which, for
example, takes place in nucleic acid diagnostics or CL ELISA
applications (where AEs are used as CL indicators) – obtaining
a high usefulness of CL reagents at pH = 8 would be beneficial.
In this context, the XAEs containing two halogen atoms in
both ortho-positions – in particular the compound 2,6-diF
(Fig. 3B, light bars) – would be beneficial.
The previously reported trend among acridinium chemi-
luminogenic salts indicated that, in general, the dynamics of
light emission increases as the pKa of the leaving group
decreases, i.e. the moiety detached upon CL becomes a
“better” leaving group.42 In the present work we were unable
to observe a clear relationship between kinetics or efficiency
and pKa of the respective phenol among all the investi-
gated XAEs. However, in the sub-group of the salts with no
steric hindrance, i.e. in the series of 2- or 4-halosubstituted
esters, an interdependence between CL decay kinetics con-
stants (kCL) and pKa values of the respective phenols was
observed (Fig. 4).
The influence of surfactants on chemiluminescence. The
cationic, zwitterionic and neutral surfactants (CTAC, DDAPS
and Triton X-100, respectively) added to aqueous CL systems,
containing XAEs, generally increase both the dynamics as well
as the efficiency of emission of light. The parameters quanti-
tating this behavior are ΔkCL and ΔRCLE, respectively and the
relevant data are summarized in Table 3. The acceleration of
CL kinetics decay for the investigated XAEs obeys the order
(average values of ΔkCL are given in parentheses): DDAPS (1.5) >
CTAC (1.1) > Triton X-100 (0.9). The general trend observed
in all cases (CTAC, DDAPS, Triton X-100) is that the values of
ΔkCL increase with the increase of the inductive constants of
substituents present in the benzene ring (σI = 0.76, 0.52, 0.47,
0.50 and 0.39 for NO2, F, Cl, Br and I, respectively).45 On the
other hand, the enhancements of CL efficiency for the investi-
gated XAEs are arranged in the following order (average values
of ΔRCLE are given in parentheses): DDAPS (2.2) ≈ Triton
Table 3 Effects of surfactants on CL parameters for selected XAEs in alkaline aqueous environments. For details see the Experimental section
CTAC
DDAPS
Triton X-100
Compound
code
ΔkCL
Δ(RCLE)b
LOD/LOQc × 10−11
M
ΔkCL
Δ(RCLE)
LOD/LOQ × 10−11
M
ΔkCL
Δ(RCLE)
LOD/LOQ × 10−11
M
a
2-F
2-I
2-NO2
2,6-diF
2,6-diCl
2,6-diBr
2,6-diI
1.14
1.15
1.24
1.18
1.09
1.04
1.10
2.42
1.89
2.12
1.84
1.37
1.22
1.26
4.60/7.47
7.85/12.7
2.93/4.78
1.29/2.13
7.16/11.3
6.73/10.7
15.0/24.0
1.21
1.23
1.37
2.02
1.62
1.43
1.79
2.72
2.35
2.36
3.15
2.07
1.85
1.16
9.10/14.8
11.0/17.9
7.24/11.8
3.05/5.14
27.7/44.3
16.9/26.9
14.3/22.9
0.81
0.80
1.00
1.24
1.20
0.77
0.49
3.19
2.02
2.45
2.40
2.47
1.80
0.93
10.7/17.4
19.6/31.5
6.36/10.2
7.28/11.8
17.1/27.2
14.5/23.1
17.3/27.5
Abbreviations: a ΔkCL – denotes the change of CL decay kinetic constant value relative to the value obtained with no surfactant using the
standard triggering system (H2O2/NaOH) (see footnote 1 under Table S4 (ESI)); b Δ(RCLE) – denotes the change of integral CL intensity relative to
the value obtained with no surfactant using the standard triggering system (H2O2/NaOH) (also see Table S3 (ESI)); c LOD/LOQ – denotes the limit
of detection and the limit of quantification, respectively.
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X-100 (2.2) > CTAC (1.7). The most efficient CL systems among The products secreted upon XAEs’ chemiluminescence
the studied ones such as 2,6-diF/DDAPS or 2-F/Triton X-100,
The mixtures obtained after the completion of CL were
express more than three-fold enhancement of CL (Table 3,
immediately subjected to RP-HPLC analyses in order to reveal
columns 6, 9) in comparison with the typical environment,
the type and population of the products. An exemplary chro-
in which the emission of XAEs is triggered (Table S4,
matogram for the reaction mixtures is presented in Fig. S4
column 6, ESI†).
(ESI†). The products found after CL of XAEs were always
As it was observed for similar systems, the dynamics and
10-methyl-9-acridinone (NMAON) (the most intense signals were
efficiency of CL are distinctly accelerated in the presence of
at 254 nm and 268 nm), 10-methylacridinium-9-carboxylic acid
cationic or zwitterionic surfactants, such as CTAC and
(NMACA) and the substituted phenol (RPhOH). The above
DDAPS.24,43 Such a bimolecular reaction can undergo micellar
signals were attributed by a comparison of their retention
times (RT) with the ones found for previously prepared stan-
catalysis, based on the ability of the surfactants to bind
various substrates and attract oppositely charged entities
dard substances (NMAON, NMACA)17 or those purchased
towards their surfaces.44 In the context discussed here, the
(RPhOHs) as reference compounds. The other trace signals
latter phenomenon is presumably manifested by the local
observed in the chromatograms were analytically insignificant
increase in the concentration of the hydroperoxide ions at the
and they were therefore not quantified. The data concerning
oppositely charged micellar surfaces and, resultantly the more
the content of NMACA in post-CL mixtures, assayed for com-
efficient formation of the adequate hydroperoxide adducts
pounds 2,6-diF, 2,6-diCl, 2,6-diBr and 2,6-diI under standard
CL conditions are summarized in Table 4 (column 2). As the
(Scheme 2, a) that initiate the “light path” of the reaction
(Scheme 2, pathway I). In contrast to the above, when using a
emissions from the above haloesters represent flash-type
neutral surfactant such as Triton X-100, no impact (or even a
decays, the content of the acid, assessed with the use of
slight decrease) of the rate of CL was detected – which is
calibration graphs (Fig. S4B, ESI†), is generally low and falls in
consistent with the previous observations made on similar CL
the range of ca. 0.2–2.7 mol%. The results indicate that, in the
group of investigated compounds, the content of acid in the
reaction mixtures does not clearly depend on the type of sub-
stituent. Interdependences were observed among the pseudo-
first order kinetic constants of the alkaline hydrolysis of 2,6-
disubstituted XAEs at pH = 12 (kOH) and the size parameters,
systems.24,43 However, a clear influence of the above neutral
detergent on the efficiency of emission is observed in the case
of the herein considered group of compounds, which differen-
tiates the behavior of XAEs from other acridinium esters
(labels and their conjugates) investigated in this context.24 It
should be noted that the chemiluminogenic adduct, from
characterizing the substituents (halogen atoms) (Table 4,
which the transformations start, is neutral in the case of the
columns 4 and 5). According to this, kOH takes the values
molecular systems studied here (Scheme 2, a). Taking into
which are approximately 1.0, 0.2 and 0.06 for 2,6-diF, 2,6-diCl
account the structure of Triton X-100, which is similar to the
and 2,6-diBr, respectively.
polyethylene glycols (PEGs), one can expect the occurrence of
The ESI-MS mass spectra obtained for the mixtures under
specific intermolecular interactions of the latter intermediate
CL or just after the completion of CL reaction with the partici-
(a) with this neutral detergent – such as hydrogen bondings
pation of selected XAEs in an alkaline aqueous environment
are given in Fig. 5. It can be seen that, instead of the cations
and the hydrophobic interactions of the XAEs’ aromatic ring
systems with the micelles. The latter phenomenon can lead to
the increased local concentration of the chemiluminogenic
adduct, and – resultantly – an increase in emission efficiency
(ΔRCLE) and, at the same time, diminution of the reaction
rate due to the involvement of a in the above mentioned inter-
actions. Our further investigations of this interesting problem
Table 4 Chromatographically (RP-HPLC) assessed content of XAEs’
alkaline hydrolysis product, 10-methylacridinium-9-carboxylic acid
(NMACA) in the post-CL reaction mixtures. Pseudo-first order kinetics
constants of XAEs’ alkaline hydrolysis at pH = 12 (kOH). For details see
are in progress.
For the above discussed enhanced CL systems, we also
established the limits of detection (LOD) and limits of quantifi-
cation (LOQ) at the maximal detector’s sensitivity (Table 3,
columns 4, 7, and 10). The above parameters, depending basi-
cally on the slope of the calibration graphs, the deviation of
the measured points from the linear regression, and the
number of measured points (eqn (S1) and (S2), ESI†) are of
great analytical importance.46 The relationships used for the
calculation of LOD/LOQ and the exemplary calibration graphs
are presented in Fig. S3B (ESI†). Among the studied CL
systems, the lowest values of LOD/LOQ – falling at the level of
1.3–2.9 × 10−11 M for LOD and 2.1–4.8 × 10−11 M for LOQ –
were obtained with the participation of 2,6-diF and 2-NO2 and
a cationic surfactant CTAC.
the Experimental section
Charton’s size values
(σV)/Taft size parameter
(ES) for adequate
NMACA
Compound contenta
CV
(%)
kOH
b
code
(mol%)
(×10−2 s−1
)
halogen atom [H]
2,6-diF
2,6-diCl
2,6-diBr
2,6-diI
0.18
1.66
2.73
1.07
0.0
3.8
5.9
4.9
4.69
0.89
0.31
—
0.27/−0.46
0.55/−0.97
0.65/−1.16
0.78/−1.40
a Signals were identified by the comparison of their retention times
with the ones assessed for the reference material. Values were
established based on the areas under the signal of NMACA in
relationship to the calibration plots (Fig. S4 (ESI)). b Values were
obtained upon the linearization of ln Ct/C0 = f(time) interdependences
(n = 6–8, R2 = 0.97–0.99) (Fig. S5 (ESI)).
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Fig. 5 ESI-MS spectra recorded in the positive ion regime for exemplary XAEs (Scheme 1). (A) 9-[(2,6-difluorophenoxy)carbonyl]-10-methyl-
acridinium triflate (2,6-diF); (B) 9-[(2,6-diiodophenoxy)carbonyl]-10-methylacridinium triflate (2,6-diI). For details, see the Experimental section.
observed in the MALDI-TOF experiments (Fig. S2 (ESI†)), of several percents (ca. 15 and 27% for 2,6-diF and 2,6-diI,
signals arise from the charged products of the oxidative respectively).
decomposition of the acridinium salts, i.e. m/z ≈ 210 (NMAON)
and m/z ≈ 238 (NMACA). The relative abundance of the latter
cation is traceable in the case of the investigated 2,6-disubsti-
tuted salts (2,6-diF and 2,6-diI) – which underlines the state-
ments drawn from the above-discussed RP-HPLC experiments.
Conclusions
It should also be noted that, among the cations detected, an
acridinium fragment corresponding to the detachment of the
substituted phenoxy fragment from the parent acridinium
cation (m/z ≈ 221 (M − 1)) has a relative abundance at the level
Newly synthesized acridinium esters containing substituents
of various electronic and steric characters such as halogens,
CF3, NO2, and OCH3 located at the ortho-, para- or meta-
positions of the benzene ring (XAEs, Scheme 1) were
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subjected to
a
comprehensive theoretical and physico-
The measurements of the XAE time profiles enabled us to
chemical investigation. The main tasks of this work assess the CL decay constants (kCL) and the Relative Chemilu-
included the assessment of the mechanism of their chemi- minescence Efficiencies (RCLE) in various environments
luminogenic oxidation in basic protic solvents, as well as a (Table S4, ESI†). To do so, we tested XAEs in aqueous or
thorough evaluation of their emission abilities in various methanolic solutions, containing various bases and additives
environments.
such as sodium hydroxide, tetrabutylammonium hydroxide
By applying computational methods, based on Density and 1,8-diazabicyclo[5.4.0]undec-7-ene, as well as cationic,
Functional Theory (DFT), we have proposed a number of poss- zwitterionic or neutral surfactants such as cetyltriethyl-
ible steps concerning the transformations of XAEs in an ammonium chloride, dimethyldodecylammonio-1,3-propane
aqueous alkaline environment, with the participation of ions sulfonate and Triton X-100. All the XAEs, except 2-OCH3 (the
such as OH− and OOH−. The latter may compete for the elec- only chemiluminogenic salt in the studied group, bearing an
tron-deficient acridinium C(9) atom in the initial steps of the electron-donor substituent) emit a flash-type blue radiation
XAE transformations. In general, three concurrent reaction (ca. 450–455 nm) in aqueous or methanolic solutions, completed
pathways have been proposed. Pathway one (blue arrows, within 1–2 seconds, regardless of the steric hindrance caused
Scheme 2) comprises the “light way” of XAE oxidation, invol- by spatial atoms (Br, I) attached at the ortho positions of the
ving 1.7–2.4% of the reaction energy released upon the trans- lateral benzene ring of XAEs. In all cases the aqueous trigger-
formation with regard to experimentally obtained values of ing reagents, including quaternary ammonium base (H2O2
+
quantum yields (QY, Table S4 (ESI†)). As stated before,22,33 CL TBAOH) allowed obtaining 2–20% higher RCLE if compared to
of the acridinium cations starts from the attack of OOH− on standard (H2O2 + NaOH) or other triggers (H2O2 + DBU; H2O2
the electron-deficient C(9).22,33 Interestingly, the acridane-type + TBAOH in CH3OH) (Fig. 2B). A distinct decrease in the XAEs’
adduct thus formed undergoes further transformations via two RCLE values upon lowering of the aqueous environment’s pH
slightly different channels – suggesting thermodynamic fea- was observed (Fig. 3A). The effect was most pronounced in the
tures of the investigated systems and the values of the pz case of the 2-OCH3 (deactivated by the substituent for the
LUMO orbital at the acridinium C(9) atom, reflecting suscepti- nucleophilic attack) while it is less pronounced in the cases
bility of XAEs for the attack of a nucleophile. The two alterna- where the esters bear substituents of a distinct negative induc-
tive pathways seem to be dependent on the character of the tive effect, such as 2-NO2 or 2,6-diF (retaining ca. 50% of emis-
“leaving group”, being detached upon the reaction. The fluori- sion ability at pH = 8). Among the compounds investigated,
nated esters such as 2-F, 2-CF3 and 2-methoxy (2-OCH3) are 2-OCH3, 4-Br and 3-F can be recommended as fragments of CL
characterized by the occurrence of the cyclic dioxethane labels due to their high usefulness at pH = 12 (the product of
anionic form (b, Scheme 2) at the “bottleneck” stage of the relative efficiency and stability at given pH), while compounds
pathway and behave similarly to the previously investigated 2,6-diF, 2-NO2, expressing high usefulness at pH = 8, are inter-
alkyl-acridinium esters.22 The second pathway includes the for- esting reagents in applications where high alkalinity of the
mation of the cyclic dioxethanone – the structure suggested in environment makes a limitation (Fig. 3B).
the literature as the crucial intermediate of the acridinium
The screening of the influence of surfactants on the emis-
ester CL transformations – although till now there is no clear sion ability and kinetics enabled us to select the CL systems of
experimental evidence demonstrating its occurrence. Both optimal performance – some of them, containing pairs of
cyclic intermediates make an immediate, high-energetic pre- reagents such as 2,6-diF/DDAPS or 2-F/Triton X-100, express
cursor of the electronically excited molecules of 10-methyl-9- more than 3 times higher efficiency compared to the emission
acridinone (NMAON), the final emitter in all the CL systems in typical aqueous environments with no enhancers (H2O2/
based on acridinium salts.17,47
NaOH).
The amounts of the acid (NMACA), the product of XAEs’
The other two general pathways are considered as “dark” –
making concurrent reaction channels to the above-discussed alkaline hydrolysis, detected in the post-CL reaction mixtures,
“light way”. The latter two pathways denote a “pseudobase are traceable (below 2.5 mol%). The latter presumably arise
way” (pathway two, red arrows, Scheme 2) – ending with the from the fact that the values of alkaline hydrolysis constants
formation of NMAON in an electronic ground state and a (kOH) are 2–3 orders of magnitude lower than the respective
“hydrolytic way” (pathway three, green arrows, Scheme 2) – constants of CL decay (kCL), as revealed among acridinium 2,6-
ending with the formation of 10-methylacridine-9-carboxylic dihaloesters (Table 3 and S3 (ESI†)). Other experiments
acid (NMACA) in the ground electronic state. Each of the three revealed that the alkaline hydrolysis at pH = 12 of sterically
pathways ends up with chemically stable products, which were hindered compounds, such as 2,6-diBr, occurs ca. 17 times
observed experimentally using chromatographic (RP-HPLC) slower than the hydrolysis of the compound 2,6-diF, as indi-
and mass spectrometry (ESI-MS) techniques. Their population cated by values of kOH (Table 4).
in the reaction mixtures upon the completion of CL reflects
We hope the present work will enhance the knowledge on
the character of the substituents present in the benzene ring the mechanism of oxidative transformations and physico-
of the XAEs. However, at this stage of the research, we were chemistry of the acridinium esters, bearing various atoms or
unable to observe any of the high-energetic reaction inter- groups of atoms in their phenoxy moieties, involved in the
mediates revealed by computations.
generation of light. Knowledge on their CL properties in
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various liquid environments is useful in the rational design of Ionization Time of Flight) as well as ESI (Electrospray Ioniza-
new luminogenic labels and indicators to be employed in tion) mass spectrometry (MS) techniques. The nuclear mag-
ultrasensitive biomedical and/or environmental analytics.
netic resonance spectra (1H NMR, 13C NMR) were registered in
CD3CN, CD3OD or DMSO-d6 (300 K) using a Unity 500 Plus
spectrometer (Varian) with a 5 mm PFG probe. In some cases,
the two-dimensional 1H–1H COSY and HETCOR techniques
were employed for the unequivocal assignment of signals
(compounds 2-F, 2-Cl, 2,6-diBr and 4-Cl). The experimental
results were compared with the simulated spectra generated
Experimental
Computations
Unconstrained geometry optimizations of isolated molecules
(Scheme 1) were carried out, at the DFT level of theory,48 using
the B3LYP functional49 and 6-31G** basis sets50 implemented
in the GAUSSIAN 09 program package.51 After completion of
each optimization, the Hessian (second derivatives of the
energy as a function of the nuclear coordinates) was calculated
to assess whether stationary structures had been obtained.48
The harmonic vibrational frequencies were then derived from
the numerical values of these second derivatives and used to
obtain the enthalpy and Gibbs’ free energy contributions at
298.15 K and standard pressure with the aid of a built-in com-
putational program of statistical thermodynamics routines.52
The solvent (water) effect with complete optimization of each
molecule was included in the DFT calculations at the level of
the Polarized Continuum Model (PCM) (UAHF radii were used
to obtain the molecular cavity).53
The enthalpies (Δr,298H°) and Gibbs’ free energies (free
energies in the case of DFT(PCM)) (Δr,298G°) of the reactions
(r), as well as the enthalpies (Δa,298H°) and Gibbs’ free energies
(free energies in the case of DFT(PCM)) (Δa,298G°) of activation
(a) indicated in Scheme 1 were calculated by following the
basic rules of thermodynamics.54 The rate constants (298k°) for
the gaseous phase reactions were obtained by applying the
equation:
1
with the aid of the H NMR ACD package.55 The masses of the
parent cations involved in XAEs were established using a VG
Trio-3 triple quadrupole mass spectrometer (Masslab, Altrinc-
ham, UK) with 2,5-dihydroxybenzoic acid (DHB) as a matrix.
Mass spectra were acquired in the positive polarity mode with
the 100–700 m/z range of scan. The laser was set to 125 shots
and the TOF detector was set to a linear mode with a 20 kV
difference of potentials. The molecular ions recorded, together
with basic fragmental ions, are summarized in Table S1 (ESI†).
In the case of selected XAEs, ESI-MS spectra were registered for
the post-CL reaction mixtures, using a Bruker-Esquire LC Elec-
trospray Ionisation Mass detector (Bruker-Daltonic GmbH,
Germany). The mass spectra were acquired in the positive ion
mode in the scan range of m/z 100–600. Capillary exit, trap
drive and skimmer voltages were set to 81.9 V, 27.6 V and 15 V,
respectively.
Measurement of chemiluminescence
Investigations of XAEs’ (Scheme 1) chemiluminescence were
conducted in two fashions using a Fluoroskan Ascent FL plate
luminometer (Labsystems-Thermo). To compare the kinetics
and efficiency of the emission, the equipment was operated at
moderate sensitivity (photomultiplier tube (PMT) voltage =
340 mV) and a resolution of 20 ms per point. To record the
kinetic profiles, the compounds were dissolved in anhydrous
acetonitrile (Aldrich) to obtain a concentration of 5.0 mM. Por-
tions of such prepared stock solutions were diluted with 1 mM
aqueous HCl to obtain a concentration of 4 × 10−5 M and
200 μl aliquots of the latter were serially dispensed (n = 5) over
a 96-well white polystyrene plate (Cliniplate). Next, portions of
H2O2 solution (100 μl, 0.06%) in 0.01 M HNO3 were added to
each well and the plate was shaken for 30 seconds. CL was trig-
gered by adding 100 μl of NaOH solution (0.2 M) and collect-
ing the light output over 2-minute periods at 298 K. The above
experiments were repeated in the same manner using other
RT
Nh
298k° ¼
exp½ꢀΔa;298G°=ðRTÞꢁ
ð1Þ
resulting from the transition state theory, and the reaction
completion time (298τ99) from the formula:
298τ99 ¼ ln 100=298k°
ð2Þ
where R, T, N, and h denote the gas constant, temperature
(298.15 K), Avogadro number, and Planck’s constant,
respectively.54
Chemical identity of XAEs and spectroscopic analyses of XAEs
9-[(Phenoxy)carbonyl]-10-methylacridinium triflates, substi- bases (c = 0.2 M): tetrabutylammonium hydroxide (TBAOH)
tuted in the benzene ring (XAEs, Scheme 1), were synthesized and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Values of CL
by adopting the procedures reported before.25–32 The struc- decay constants (kCL) were calculated as negative values of the
tures and IUPAC names of the compounds obtained for this slopes of ln(ICL/I0CL) = f (time (s)) dependences.
work are depicted in Scheme 1. The detailed data, concerning
To measure the efficiency of CL, the above acidic solutions
chemical synthesis and identity (retention times (RP-HPLC), of XAEs were serially diluted with 1 mM HCl to obtain solu-
melting points, elemental analysis) of the compounds investi- tions in the range of concentration 4 × 10−5–4 × 10−8 M and
gated in this work are given in the ESI.†
were treated as described above. The integral light outputs
XAEs were subjected to structural analyses with the aid of were collected by calculating the area under each kinetic
nuclear magnetic resonance spectroscopy (1H NMR, 13C NMR, profile, applying the implemented Ascent FL software. The
1H–1H COSY) and MALDI-TOF (Matrix Assisted Laser Desorption calibration graphs were obtained upon linearization of the fol-
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lowing relationship: Area under CL profile (A, RLU2) = f(XAE Products of XAEs’ chemiluminescence
concentration (M)). The values of emission efficiency for each
The products of CL were assayed directly after completion of
the emission of light with the aid of a Waters chromatographic
system (see the ESI†). For these experiments, the column
employed was a 150 × 4.60 C6 Phenyl 110, 5 µm (Gemini,
Phenomenex) and the mobile phase consisted of 60%/40% v/v
acetonitrile/phosphate buffer (2 mM Na2HPO4 + NaHPO4,
pH = 3.5). The emission of XAEs was triggered in Eppendorf
vials, by mixing 20 µL of XAE (5 mM solution in anhydrous
acetonitrile) with 50 µl of H2O2 in 0.1 M HNO3, followed by the
addition of 0.2 M aqueous NaOH (50 µL). After completion of
the emission, the mixtures were acidified with a drop of H3PO4
and immediately subjected to chromatographic analyses
(absorption detection at 254 and 268 nm). Post-CL reaction
products were identified by a comparison of their retention
times (RT) and signal intensities (A) with the analogous para-
meters obtained for standard substances – namely 10-methyl-
9-acridinone (NMAON), 10-methylacridinium-9-carboxylic acid
(NMACA) and the respective phenols (RPhOHs): 2,6-difluoro-
phenol, 2,6-dichlorophenol, 2,6-dibromophenol and 2,6-di-
iodophenol. Calibration graphs prepared for NMAON and
NMACA enabled the assessment of the content of the latter
product in the post-CL reaction mixtures (Table 4, Fig. S4†).
To assess the pseudo-first order kinetic constants of alka-
line hydrolysis for selected XAEs (kOH, Table 4), the XAEs were
dissolved in phosphate buffer (pH = 12, cXAE = 1.0–1.5 mM)
and were incubated at 298 K for up to 5 min. To stop the reac-
tion after a certain time, 200 µl of each of the above mixtures
were treated with 10 µl solution of the TFA/mobile phase
(1/3 v/v) to obtain a final pH = 3. Hydrolysis constants (kOH) were
established, using the above-mentioned Waters HPLC system
(mobile phase: acetonitrile/0.1% TFAaq = 1/1 v/v; stationary
phase: Waters Symmetry C8, 4.6 × 250 mm, 5 μm) with the UV-
absorption detector set at 256 nm. Chromatographic runs were
recorded after specified reaction times (20–200 s, n = 3) and
the areas under the NMAON and NMACA signals were calcu-
lated using built-in Empower™ software. The values of kOH
were established graphically, using a linear calibration chart,
assuming c0 values as the initial concentration of XAE in the
mixture and ct values as the temporary concentration of the
hydrolysis product (NMACA), established according to the
areas under the signal (Fig. S5 (ESI†)).
CL system were obtained as the slopes of the linear calibration
graphs and normalized to the highest value (RCLE).
To compare the CL efficiencies of XAEs at various pH, 50 μl
aliquots of their acidic solutions (c = 4 × 10−5 M) were added to
the wells of the white plate (n = 5). Next, a 50 μl portion of
0.12% w/w H2O2 in 0.01 M HNO3 was added to each well and
the plate was shaken for 30 seconds. CL was triggered by
adding 200 μl Britton-Robinson buffer (pH = 8.0, 10.0, 12.0)
and the integral light outputs (A) were collected over 2-minute
periods. The CL efficiency for XAEs was calculated from the
formula:
CL efficiency ¼ 100% ꢂ A; ðgiven pHÞ=A
for the most efficient system:
To assess the stability of XAEs in alkaline environments,
XAE stock solutions were diluted with Britton Robinson buffer
(pH = 8.0), to reach a final concentration of salt of 4 × 10−5
M. The solutions were then incubated at 298 K for 30 minutes
and 200 μl aliquots of the latter mixtures were added to each
well of the plate (n = 5), followed by the addition of 100 μl of
0.06% H2O2 in 0.125 M HNO3 (to obtain pH = 3.0–3.5) and a
30-second shaking of the mixtures. To trigger the emission,
100 μl of NaOH (0.3 M) was added (final pH = 12) and the light
outputs were integrated over 2 minute periods. The stability of
XAEs, regarded as the preserved initial activity, was calculated
from the following equation:
Stability ¼ 100% ꢂ A ðt ¼ 30 min: incubationÞ=A
ðt ¼ 0; no incubationÞ:
A second type of CL experiment was performed at the
maximal sensitivity of the apparatus (PMT voltage = 1000 mV)
for selected XAEs in the presence of various surfactants:
cetyltrimethylammonium chloride (CTAC), dimethyl-
dodecylammoniumpropane sulfonate (DDAPS) and Triton
X-100. The acridinium salts were serially dissolved in 1 mM
HCl to obtain a series of solutions of concentrations in the
range of 5.0 × 10−11−2.5 × 10−9 M. The solutions of surfactants
(24 mM of CTAC or DDAPS and 60 mM of Triton X-100) were
optimized and prepared in the oxidizing solution (0.01 M
HNO3 containing 0.06% H2O2). 100 μl aliquots of XAE solu-
tions were distributed over a 96-well, white polystyrene plate
(n = 5), followed by the addition of 50 µl of the solution of the
Acknowledgements
oxidant containing each of the above surfactants. CL was trig- This work was financed by the State Funds for Scientific
gered by the addition of 50 µl of 0.2 M KOH to each well. Tem- Research through grant no. N N204 375740 (contract no. 3757/
porary intensities of CL (ICL, RLU) were collected every 20 ms B/H03/2011/40) for the period 2010–2013 and by the National
until the reactions were complete (up to 2 minutes). For com- Scientific Center (NCN) through grant no. UMO-2012/05/B/
parison, the measurements were taken under the same con- ST5/01680 for the period 2013–2016. B.Z. acknowledges finan-
ditions with no surfactants. The changes in the values of CL cial support from the European Social Fund within the project
decay kinetic constants (ΔkCL) and emission efficiencies “The development program of the University of Gdansk in
(ΔRCLE) were calculated as the ratio of the value obtained areas of Europe 2020 (UG 2020)”. Calculations were carried out
under experiments carried out in the presence of a surfactant on the computers of the Tri-City Academic Network Computer
and the adequate value obtained with no surfactant (Table 3).
Centre (TASK) and the Wroclaw Centre for Networking and
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Supercomputing (WCSS) (grant no. 215). The authors would 16 K. A. Browne, D. D. Deheyn, G. A. El-Hiti, K. Smith and
like to thank Dr Anna Białk-Bielińska and DSc Marek Gołę- I. Weeks, J. Am. Chem. Soc., 2011, 133, 14637–14648.
biowski for recording the mass spectra. We are greatly 17 K. Krzymiński, A. D. Roshal, B. Zadykowicz, A. Białk-
indebted to Dr Dorota Zarzeczańska for her contribution to
the pH measurements and Mrs Kinga Zelewska for her contri-
Bielińska and A. Sieradzan, J. Phys. Chem. A, 2010, 114,
10550–10562.
bution to the preparation of samples for spectroscopic 18 A. Natrajan, Q. Jiang, D. Sharpe and J. Costello, High
analyses.
quantum yield acridinium compounds and their uses
in improving assay sensitivity, United States Patent No
US7,309,615B2, publ. date: Dec. 12, 2007.
19 R. Renotte, G. Sarlet, L. Thunus and R. Lejeune, Lumine-
scence, 2000, 15, 311–320.
20 A. Natrajan, Q. Jiang and D. Sharpe, Stable acridinium
esters with fast light emission, United States Patent Appli-
cation Publication, US2010/0099077A1, publ. date: Apr. 22,
2010.
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